Dynabeads regulatory cd4 cd25 t cell kit

Tregs were immediately purified from peripheral blood mononuclear cells, which were isolated from 100 ml of fresh heparinized peripheral blood collected from healthy volunteers by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) gradient centrifugation and by immunomagnetic separation using the Dynabeads® Regulatory CD4⁺CD25⁺ T-cell Kit (Invitrogen™, Oslo, Norway), according to the manufacturer’s instructions. The procedure yielded a highly pure preparation (> 90% purity) of regulatory CD4⁺CD25⁺ T-cells, more than 80% of which expressed the intracellular transcription factor Foxp3. The isolated Tregs were expanded with Dynabeads® Human Treg Expander (Gibco®, Oslo, Norway) containing 100 U/ml recombinant human interleukin (rIL-2) (Gibco®, Carlsbad, CA, USA). Each batch Tregs was treated with a standard protocol. After isolation, they were cultured for 10 days and expanded to more than 2 × 10⁶. Each experiment was performed by three independent batches in duplicate. The study was approved by the Institutional Review Committee of E-DA Hospital, and volunteer donors provided written informed consent.

Sex-matched, 8- to 12-week-old (B6, H-2^b) recipient mice received myeloablative total body irradiation (TBI) of 9 Gy using a Faxitron CP-160 x ray irradiation system (Faxitron X-Ray). Within 4 hours after TBI, mice were i.v. injected (retro-orbitally) with 5 × 10⁶ FVB/N T cell–depleted (TCD) BM cells for hematopoietic reconstitution. T cells from the BM were depleted using CD90.1 MicroBeads (Miltenyi Biotec) following manufacturer instructions. To induce aGvHD, allogeneic enriched 6 × 10⁵ CD4⁺ T cells from FVB/N or FVB.L2G85 mice were coinjected i.v. T cells were purified from the spleen using Dynabeads Untouched Mouse CD4⁺ Cells Kit (Invitrogen), whereas Tregs were purified from the spleen using Dynabeads Regulatory CD4⁺/CD25⁺ T Cell Kit (Invitrogen), according to manufacturer instructions. Cell purity was accessed with flow cytometry (>95% purity).

Peripheral blood mononuclear cells (PBMCs) were isolated from 100 ml of fresh heparinized peripheral blood from healthy volunteers by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) gradient centrifugation. The cells from the interface were washed three times in HBSS and seeded in 6-well microtiter plates in RPMI-1640 medium, and allowed to settle at 37 °C in a humidified atmosphere with 5% CO₂. After 30 min, the non-adherent cells were collected as lymphocytes. Treg cells were immediately purified from PBMCs by immunomagnetic separation using the Dynabeads® Regulatory CD4⁺CD25⁺ T Cell Kit (Invitrogen™, Oslo, Norway), according to the manufacturer’s instructions. In brief, CD4⁺ cells were isolated by negative selection after depletion of cells expressing CD8, CD14, CD16, CD19, CD36, CDw123, CD235a and CD56. CD25⁺ cells were then selected by positive selection using magnetic beads directly conjugated to an anti-CD25 Ab. The procedure yielded a highly pure preparation (>95% purity) of regulatory CD4⁺CD25⁺ T cells, more than 80% of which expressed the intracellular transcription factor, Foxp3. The isolated Treg cells were expanded with Dynabeads® Human Treg Expander (Gibco®, Oslo, Norway) containing 100 U/ml rIL2 (Gibco®, Carlsbad, CA, USA). The study was approved by the Institutional Review Committee of E-DA Hospital and the volunteers provided written informed consent.